Association of yeast RNA polymerase I with a nucleolar substructure active in rRNA synthesis and processing.

Abstract

A novel ribonucleoprotein complex enriched in nucleolar proteins was purified from yeast extracts and constituents were identified by mass spectrometry. When isolated from rapidly growing cells, the assembly contained ribonucleic acid (RNA) polymerase (pol) I, and some of its transcription factors like TATA-binding protein (TBP), Rrn3p, Rrn5p, Rrn7p, and Reb1p along with rRNA processing factors, like Nop1p, Cbf5p, Nhp2p, and Rrp5p. The small nucleolar RNAs (snoRNAs) U3, U14, and MRP were also found to be associated with the complex, which supports accurate transcription, termination, and pseudouridylation of rRNA. Formation of the complex did not depend on pol I, and the complex could efficiently recruit exogenous pol I into active ribosomal DNA (rDNA) transcription units. Visualization of the complex by electron microscopy and immunogold labeling revealed a characteristic cluster-forming network of nonuniform size containing nucleolar proteins like Nop1p and Fpr3p and attached pol I. Our results support the idea that a functional nucleolar subdomain formed independently of the state of rDNA transcription may serve as a scaffold for coordinated rRNA synthesis and processing.

Copurification of nucleolar proteins. (A) Fractionation scheme of WCEs. WCE was prepared and fractionated as described (Tschochner 1996; Milkereit et al. 1997). The relative amounts of total protein in the single fractions are indicated in italics. Note that <1% of the proteins were found in fraction PA600. (B) Monitoring of different proteins through the established purification scheme. 20 μg of WCE, 8 μg of fractions K90, K350, and T0, and 4 μg of fraction PA600 were separated by 10% SDS-PAGE and analyzed by Western blotting with antibodies as indicated. (C) Gel filtration of WCEs in the presence of 300 mM acetate. 0.15 ml WCE (34 mg/ml) of yeast strain YJV166 (Venema and Tollervey 1996), which contained protein A–tagged Rrp5p, was generated in buffers with low ionic strength and fractionated on a Superose-6 column (FPLC) in the presence of 300 mM potassium acetate. 0.1 ml of each fraction was TCA precipitated and analyzed by Western blotting. Nop1p, yeast fibrillarin; A190, pol I subunit A190; Rrp5p-ProtA, protein A–tagged Rrp5p; Reb1p, pol I–specific transcription termination factor. (D) Immunoprecipitation of HA-tagged A43. Equal amounts of WCEs derived from strains Ypg2, which contained a HA- and His6-tagged A43 subunit, and OG39-6d (Δ135) in which A135 has been disrupted, were immunoprecipitated with antibodies directed against the HA tag. 20 μg of WCE (lanes 1 and 2) and 10% of the immunoprecipitations (lanes 3–5) were analyzed by Western blotting. Lane 5: same conditions as lane 3; however, treatment of the WCE with a mixture of RNase and DNase precluded the immunoprecipitation. (E) Affinity purification of His6-tagged A43. 8.5 mg of fraction K350 derived from yeast strain Gpy2 that contained a HA- and His6-tagged A43 subunit and the same amount of the Δ135 strain, which contained approximately the same amount of Nop1p, were nuclease-treated (see above), loaded on Ni-agarose (0.5 ml) in the presence of buffer A supplemented with 10 mM imidazole, washed with buffer BU300 including 10 mM imidazole, and eluted with buffer BU300 including 250 mM imidazole. 0.1% of the load and 2% of the elution step was analyzed by Western blotting (upper panel). Increasing volumes of the eluate derived from the His6-A43 strain were tested in transcription initiation (pol I-A, purified pol I which lacks initiation factors).

Analysis of fraction PA600 by gel filtration on Superose-6. Coprecipitated proteins were resolubilized in buffer BU600, which contained 600 mM potassium acetate diluted to an ionic strength of 300 mM potassium acetate, centrifuged, and 50 μl was applied to a Superose-6 column (SMART; Amersham Pharmacia Biotech) and processed with a flow rate of 12.5 μl/min in buffer BU300. Fractions of 50 μl were collected and 25 μl of each was analyzed by Western blotting.

Identification of proteins of the assembly by mass spectrometry. (A) pol I present in fraction PA600 was further purified on BioRex70 and separated on a Sephacryl S-300 column in the presence of 300 mM potassium acetate as described previously (Milkereit et al. 1997). The peak fraction in terms of protein content, which also contained most of pol I (data not shown), was separated by SDS-PAGE. Single bands of silver-stained gels were excised, in-gel digested with trypsin, and analyzed by MALDI mass spectrometry followed by database searching with the resulting peptide masses. Due to high mass accuracy of MALDI peptide maps, proteins were identified unambiguously. (B) MALDI mass spectrum of peptides recovered after tryptic in-gel digestion of single protein band. A search with detected masses in nonredundant protein database showed that 18 peaks (P) matched to calculated tryptic peptide masses of yeast FK506-binding nuclear protein (Fpr3) within an accuracy better than 50 ppm and covered 33% of the sequence. The peaks marked by M correspond to tryptic autolysis products.

In vitro activities of the isolated nucleolar assembly. (A) Fraction PA600 is capable of initiating and terminating rDNA promoter-dependent transcription. 16 μg of fraction T0 and 1.25 μg of fraction PA600 that contained both approximately equal amounts of pol I and 0.8 μg of purified pol I (pol I-A) were used in promoter-dependent transcription assays. In reactions depicted in lanes 1–3, 100 ng of plasmid pSES5, which contained the rDNA promoter but did not contain the termination site, served as a template. The plasmid was linearized with EcORV before adding to the assays analyzed in lanes 2 and 3. Each 100 ng of uncut plasmid pSIRT (Musters et al., 1989b), which contained both the rDNA promoter and the termination site, was used in reactions analyzed in lanes 4–6. (B) Fraction PA600 is capable of pseudouridylating RNA. 38 U of T7-pol in the absence (panel a) or presence (panel b) of 0.3 μg of the tRNA-specific pseudouridylation factor Pus1p, 5 μg of purified pol I (pol I-p) (panel c), and 12.5 μg of fraction PA600 (panel d) were used to synthesize RNA. DNA coding for intron containing Ile-tRNA (Szweykowska Kulinska et al. 1994; Simos et al. 1996) (panels a and b), template pItailKS (Tschochner and Milkereit 1997) containing an extended 3′ end, at which purified pol I-p can initiate transcription without promoter, and plasmid pSIRT (panel d) served as templates, respectively. Transcription was performed in the presence of [32P]UTP and [32P]GTP. Labeled transcripts were separated on a 7% polyacrylamide/7 M urea gel and transcripts corresponding to the expected size were excised from the gel and digested with RNase T2. The resulting nucleotides were analyzed by two-dimensional thin-layer chromatography as described (Grosjean et al. 1990).

The isolated complex contains nucleolar RNA. WCEs and fractions generated according to the established purification scheme were prepared and analyzed by Northern blotting with specific oligonucleotides as described in Materials and Methods.

The nucleolar assembly isolated from pol I–deficient cells can recruit pol I to result in a transcriptionally active complex. (A) The nucleolar components copurify and coprecipitate when prepared from yeast strains not active in pol I–dependent transcription. (Upper panel) 8 μg of fraction PA600 derived from stationary cells (lane 3), from strain delta 135, which was disrupted in the A135 pol I subunit (strain OG39-6d) (lane 2), and from growing cells was analyzed by Western blotting with antibodies as indicated. (Lower panel) 1.25 μg of each PA600 fraction was analyzed in promoter-dependent transcription. (B) Purified pol I can be reassociated into a functionable assembly. Purified pol I-p (7 μl), which also contained pol I/Rrn3p complex but lacked TBP-cpl and fraction B600 (see Milkereit et al. 1997; Milkereit and Tschochner 1998) and thus was not able to initiate transcription on the rDNA promoter by itself, was combined with 12.5 μg (lanes 1–3) or without (lanes 4–6) fraction PA600 derived from strain Δ135 lacking A135 and adjusted to 1.5 M potassium acetate. The fractions were dialyzed against buffer BU100 containing 100 mM potassium acetate and centrifuged. Precipitated components were resolubilized in buffer BU600 containing 600 mM acetate and analyzed in promoter-dependent transcription. (C) In vitro assay to analyze the pol I–recruiting activity. Purified pol I coprecipitates with components present in fraction B2000 (Δ135) rather than with constituents in fraction B600 (Δ135). 0.5 μg pol I-A was combined with either 6 μg fraction PA600, 4.8 μg fraction B600, or fraction B2000, all derived from the pol I–deficient strain Δ135 in a total volume of 25 μl, adjusted to 1.5 M potassium acetate, dialyzed against buffer BU100, and centrifuged. Precipitates were resolubilized in 15 μl BU600 and analyzed in nonspecific RNA synthesis.

Electron microscopic analysis of the nucleolar assembly. (A and B) Clusters observed in fraction PA600 diluted 100 times in buffer B (see Material and Methods). (C and D) Clusters observed in fraction PA600 Δ135 diluted in the same conditions as above. (E) Field of fraction B2000 diluted in high ionic strength buffer. Pol I molecules are homogeneously dispersed on the carbon film (see also Schultz et al. 1990; Milkereit et al. 1997). The arrowhead points towards a pol I particle. (F) Field of fraction B2000 diluted in low–ionic strength buffer. The low ionic strength induces the formation of the clusters. The arrowheads point towards particles of the size of pol I. (G) Cluster labeled with the immunogold probe directed against Fpr3p. (H) Cluster labeled with the immunogold probe directed against Nop1p. Bar: 100 nm (A–F); and 50 nm (G and H).